Binding to and Inhibition of Insulin-Regulated Aminopeptidase by Macrocyclic Disulfides Enhances Spine Density

Abstract

Angiotensin IV (Ang IV) and related peptide analogs, as well as nonpeptide inhibitors of insulin-regulated aminopeptidase (IRAP), have previously been shown to enhance memory and cognition in animal models. Furthermore, the endogenous IRAP substrates oxytocin and vasopressin are known to facilitate learning and memory. In this study, the two recently synthesized 13-membered macrocyclic competitive IRAP inhibitors HA08 and HA09, which were designed to mimic the N terminus of oxytocin and vasopressin, were assessed and compared based on their ability to bind to the IRAP active site, and alter dendritic spine density in rat hippocampal primary cultures. The binding modes of the IRAP inhibitors HA08, HA09, and of Ang IV in either the extended or g-turn conformation at the C terminus to human IRAP were predicted by docking and molecular dynamics simulations. The binding free energies calculated with the linear interaction energy method, which are in excellent agreement with experimental data and simulations, have been used to explain the differences in activities of the IRAP inhibitors, both of which are structurally very similar, but differ only with regard to one stereogenic center. In addition, we show that HA08, which is 100-fold more potent than the epimer HA09, can enhance dendritic spine number and alter morphology, a process associated with memory facilitation. Therefore, HA08, one of the most potent IRAP inhibitors known today, may serve as a suitable starting point for medicinal chemistry programs aided by MD simulations aimed at discovering more drug-like cognitive enhancers acting via augmenting synaptic plasticity.

Synthesis, Evaluation and Proposed Binding Pose of Substituted Spiro‐Oxindole Dihydroquinazolinones as IRAP Inhibitors

“…As the zinc ion is a critical component of substrate recognition and processing during the catalytic cycle, we wondered if the space between the ligand and the Zn 2+ ion could simultaneously accommodate the small L‐Leu‐ p NA substrate used in our assays, and whether such simultaneous binding would retain discrimination between S and R stereoisomers. Superposition of the IRAP‐substrate complex with the IRAP‐ 38 equilibrated pose (Figure b) shows how the substrate is predicted to bind, placing the Leucine and para ‐nitrophenyl groups in equivalent positions to the Val 1 and Tyr 2 sidechains of Ang IV . As described below this model of simultaneous binding of inhibitor 38 and substrate L‐Leu‐ p NA prompted further experimental verification of the mechanism of action, i. e .…”

“…Our group synthesized both linear and constrained Ang IV analogues and macrocycles mimicking the N ‐terminal of oxytocin and obtained a series of inhibitors with high potency and stability . One of the compounds, HA08 (see Figure ), was recently proven to alter dendritic spine density in rat hippocampal primary cultures . However, HA08 and structurally related analogues are still peptidic in character and are foreseen to suffer from low bioavailability and a limited ability to pass the blood‐brain‐barrier.…”

Identification of Drug-Like Inhibitors of Insulin-Regulated Aminopeptidase Through Small-Molecule Screening

“…Truncated analogs of Ang IV, and N-terminal cyclizations thereof, provided highly potent macrocyclic IRAP inhibitors with K i 's down to 1.8 nM. [21][22][23][24][25] Although this set of peptidomimetic structures was found to be metabolically relatively stable and could enhance dendritic spine density in rat hippocampal primary cultures, 26 they suffer from poor permeability and hence are of limited use as in vivo pharmacological tools to further study the role of IRAP inhibition in models of cognition. The Stratikos group have utilized transition state mimicking compounds based on a zinc-chelating phosphinic group, and representatives of these compounds show excellent activity (IC 50 = 30 nM), but poor selectivity toward the closely related endoplasmic reticulum aminopeptidase (ERAP) 1 and 2.…”

“…pyrrolidine-1-carboxylate(26). A solution of 25 (300 mg, 0.73 mmol) and pyridine (0.35 mL, 4.37 mmol) in dry DCM (5 mL) under inert atmosphere was cooled to 0°C on an ice bath, and POCl 3 (0.12 mL, 1.3 mmol) was added dropwise.…”

The effect of insulin resistance on amygdale glucose metabolism alterations in experimental Alzheimer’s disease

“…Аналогичным образом ангиотензин IV влияет на обучение и память, ингибируя активность IRAP [15]. Так, было показано, что синтезированные 13-членные макроциклические конкурентные ин-гибиторы IRAP, разработанные путем имитации N-конца окситоцина и вазопрессина, увеличива-ли синаптическую пластичность при исследова-нии гиппокампа у крыс [16].…”